Process for delaying the deactivation of glutaryl amidase during catalysis with a thiol

ABSTRACT

A process is provided for delaying the deactivation of glutaryl amidase during catalysis. The amidase is, after maximum conversion of a substrate, separated off by filtration and can be reused in a subsequent reaction batch. In each reaction cycle, the amidase loses activity. The inactivation of the amidase can be delayed by bringing it into contact with a thiol during the reaction. The thiol is preferably 2-mercaptoethanol or cysteine.

The present invention relates to a process for delaying the deactivationof glutaryl amidase during enzyme catalysis.

7-Aminocephalosporic acid is of great commercial interest for theproduction of semisynthetic cephalosporin antibiotics.

The enzymatic synthesis of the antibiotic precursor 7-aminocephalosporicacid (7-ACS) is—as shown in FIG. 1—carried out in two reaction steps.Cephalosporin C is initially oxidized by action of D-amino acid oxidase(DAO) to α-ketoadipyl-7-ACS. In the next step, this compound ishydrolyzed to 7-ACS by glutaryl amidase (GAE).

The synthesis is carried out with the enzymes DAO and GAE, which aregenerally immobilized on supports and which are, after the reaction hasended, separated off from the solution of the product and can be reusedfor the next batch. However, if the catalyst is used repeatedly, theenzymes are deactivated, which is equivalent to enzyme consumption.

From the literature, it is known that structure-changing oxidations ofproteins can be suppressed by using thiol reagents, such as, forexample, 2-mercaptoethanol (P. Golini et al., Enzyme and MicrobialTechnology 17 (1995) 324-329; Int J. Peptide Protein Res. 48 (1996)532-538).

DAO, for example, can be regenerated using thiols. The flavoprotein DAOcatalyzes the stereospecific deamination of D-amino acids to thecorresponding α-keto acids and ammonium, for example, as shown in FIG.1, the conversion of cephalosporin C to α-ketoadipyl-7-ACS.α-Ketoadipyl-7-ACS decarboxylates in situ to glutaryl-7-ACS (G-7-ACS)(P. Golini et al., Enzyme and Microbial Technology 17 (1995) 324-329).In industry, the enzyme is frequently not employed in soluble form butimmobilized by binding to polymers, such as, for example,amino-alkylated polymers or oxirane-activated polymers. Thus, after thereaction, the enzyme catalyst can be separated off by filtration, thusbeing available for reuse. The immobilized DAO catalyst suffers partialinactivation. When the enzyme catalyst, which has already been usedonce, is reemployed in a further reaction under otherwise identicalconditions, the reaction time required for maximum conversion of thesubstrate is longer. This prolongation of the reaction time, whichoccurs on each reuse, is a measure of the stability of the catalyst inthe preparation process of G-7-ACS. The extent of the change of thereaction time to maximum substrate conversion, determined over aplurality of production cycles, is referred to as operational stability.The operational stability of immobilized DAO can be improved byseparating off the enzyme catalyst from the reaction mixture after thereaction by filtration, and treating it with a thiol, for example2-mercaptoethanol. DAO contains some easily oxidizable sulfhydrylgroups, predominantly as functional groups of the cysteine amino acidsof the protein. The action of 2-mercaptoethanol is based on the reducingaction of the thiol on the oxidation-sensitive sulfhydryl groups of thecysteines. The regeneration of these oxidized sulfhydryl groups resultsin a considerable improvement in operational stability. In theparticular case of DAO, regeneration has to be carried out afterseparation of the enzyme from the reaction mixture, because H₂O₂ isformed during the enzyme catalysis. As a strong oxidizing agent, thiswould inactivate any added thiol.

Addition of thiol may also result in a reduction in the activity ofproteins. This effect, too, can be explained by the presence of cysteineradicals. One example for this is aminoacylase. Aminoacylase is adimeric enzyme having one Zn²⁺ atom per subunit. Each subunit of theenzyme contains two cysteine SH groups and two disulfide bonds. Thechemical modification of the SH groups, such as the breaking of thedisulfide bonds, can result in an inactivation of the enzyme. It hasbeen demonstrated that the activity of aminoacylase is reduced byaddition of 2-mercaptoethanol, whereas, when the 2-mercaptoethanol isremoved by dialysis or gel filtration, the original enzyme activity canbe reestablished almost completely (W. Kördel and F. Schneider, Biochem.Biophys. Acta 445 (1976) 446-457).

Since the action of the thiol is apparently mediated by the oxidation orreduction of sulfhydryl groups of cysteine radicals, the addition ofthiols to enzymes which are known not to contain any cysteine radicalsshould consequently not result in any change of the enzyme activity.

During repeated use of GAE in catalytical conversions of the catalyst,the enzyme is, as described at the outset for the synthesis of 7-ACS(cf. FIG. 1), deactivated, which corresponds to a consumption. Thestability of the catalyst correlates with important production costs ofthe process, such as the time required, the waste produced, and thecosts of the catalyst. A process that is comparable to the processdescribed above for DAO and which is suitable for stabilizing GAE orincreasing its operational stability has hitherto not been disclosed.The enzyme GAE comprises two peptide chains (protein A and B). Theinteraction of the chains is via hydrogen bonds and hydrophilic andhydrophobic interactions of protein domains.

It is an object of the present invention to provide a process fordelaying the deactivation of glutaryl amidase during enzyme catalysis.

As can be seen from the amino acid analysis (Table 1), GAE lacks theamino acid cysteine. Thus, the use of thiol reagents for stabilizationshould not result in any enhanced operational stability of thisbiocatalyst. Contrary to expectation, it was possible to experimentallyprove the opposite. In the case of GAE, the addition of variousthiol-containing reagents, for example 2-mercaptoethanol or cysteine,resulted in a drastic increase in operational stability, depending onthe concentration.

Accordingly, the object of the present invention is achieved by aprocess for delaying the inactivation during enzyme catalysis of GAE,comprising bringing the enzyme into contact with at least one thiol. Theenzyme can optionally be present in free or supported form. A preferredsupport is, for example, an oxirane-activated polyacrylate.

Thiol or mercaptan is understood as meaning a chemical compound such as2-mercaptoethanol, glutathione, or the amino acid cysteine, the commonfeature of which is that they contain a thiol group (—SH) in themolecule.

Supported GAE consists of the enzyme catalyst GAE which is attached, forexample, to oxirane-activated or else amino-alkylated polyacrylates(=supports). A process for preparing supported GAE is described in D.Bianchi et al., Enzyme and Microbiological Technology 20 (1997) 368-372.Examples of supports are EUPERGIT® (oxirane containing polymer),AMBERLITE® XAD7 (nonionic polymeric adsorbent), and DUOLITE® A365(ion-exchange resin)(all from Röhm and Haas).

EXAMPLES

A practical application of enzyme catalysis with GAE is the preparationof 7-ACS from G-7-ACS (cf. FIG. 1). The enzyme catalysis was carried outas follows:

The substrate G-7-ACS was, in a concentration of 40 mM, reacted at 40°C. and pH 8.3 in a reaction volume of 120 ml with the immobilized GAE(from Pseudomonas diminuta). With the aid of an automated peripheralsystem, it was possible to measure process-relevant parameters (pH,temperature) on-line and to control them using a suitable controlsystem. Thus, the pH shift into the acidic range, which occurred as aresult of the reaction, was neutralized by autotitration with base. Therate of the addition of base correlated with the rate of reaction andcould be used as a measure of the degree of conversion of the substrate(G-7-ACS). PC-assisted data processing permitted the on-linedetermination of the conversion and gave the termination criterion ofthe reaction. The reaction was terminated when maximum conversion wasreached. That maximum conversion had been reached became apparent inthat it was no longer necessary to add base. Once maximum conversion hadbeen reached, the catalyst was filtered off from the solution of theproduct, and fresh substrate solution was then added. The reaction timesof the individual successive reactions (=batches) could be plotted as afunction of the batches. Via linear regression, a gradient (=Xcoefficient (min/batch)) was obtained which could be used as a measureof catalyst deactivation. The lower this value, the higher theoperational stability of the catalyst.

In one embodiment of the invention, the thiol used is 2-mercaptoethanol.This is preferably employed in a concentration range of 1 to 100 mM.

In another embodiment of the invention, it is possible to add, as thiol,cysteine, preferably in the range 1 to 100 mM.

The thiol can be used continuously, i.e., over the course of thereaction, or batchwise, i.e., in between the enzymatic reactions. Thesuccessful use can be seen from the reduced reaction time required forreaching quantitative conversion of the substrate.

The concentration of the substrate G-7-ACS was, for example, varied in arange between 5 and 500 mM. It was preferably employed in aconcentration range of from 20 to 80 mM. Over wide ranges, the result ofthe reaction was in principle independent of the reaction volumeemployed.

The use of a thiol according to any of the processes described ispossible to delay the deactivation of GAE.

Example 1 Reaction Time to Quantitative Conversion as a Function of theBatch Number without or with Cysteine

In a plurality of test series, cysteine as thiol reagent was added invarious concentrations to the substrate solution, and the reaction wascarried out as described. As can be seen from the table below, thegradient and thus, the X coefficient, decreased with increasing cysteineconcentration. Assuming linear deactivation over the reactions carriedout, it was possible to determine, for each series of measurements, thenumber of batches possible until the reaction time doubled. The valuesare shown in the table below. A considerable increase in operationalstability is evident in those cases where the reaction batchadditionally contained cysteine, and by the increase in the number ofindividual reaction batches which could be run until the reaction timeto maximum substrate conversion had doubled.

The reaction parameters were chosen as follows:

Reaction parameters: V_(R) = 120 ml T = 40° C. pH = 8.3 (Ammonia 1.66%)Amount (G-7-ACS) = 40 mM (KPP 100 mM) Amount(Cysteine)_(in the substrate) = variable Amount (GAE) = 500 U

Table for Example 1: Reaction time to quantitative conversion as afunction of the batch number without or with cysteine with with withwith without cysteine, cysteine, cysteine, cysteine, cysteine 1 mM 6 mM8 mM 10 mM X coefficient 0.414 0.131 0.077 0.046 0.016 (min/batch)Number of 36 114 195 326 938 batches until the reaction time had doubled(for linear deactivation) (−)

Comparison of the X coefficient (i.e., the increase in time per batch)for reaching quantitative conversion or the number of batches until thereaction time had doubled, as a function of the cysteine concentrationin the substrate solution

Example 2 Reaction Time to Quantitative Conversion as a Function of theBatch Number without or with Mercaptoethanol

The use of 2-mercaptoethanol as thiol reagent leads to an increase inthe operational stability of GAE. The table below shows the comparisonof the test runs with and without 2-mercaptoethanol. With respect to thestabilization of operational stability, the results for2-mercaptoethanol are comparable to those of cysteine.

The reaction parameters were chosen as follows:

Reaction parameters: V_(R) = 120 ml T = 40° C. pH = 8.3 (Ammonia 1.66%)Amount (G-7-ACS) = 40 mM Amount (GAE) = 500 U Amount (Mercaptoethanol) =1 μl/ml

Table for Example 2: Reaction time to quantitative conversion as afunction of the batch number without or with mercaptoethanol withwithout mercaptoethanol, mercaptoethanol 1 μl/ml X coefficient 0.44 0.08(min/batch) Number of batches until 41 225 the reaction time had doubled(for linear deactivation) (−)

Comparison of the X coefficient (i.e., the increase in time per batch,i.e., the gradient) for reaching quantitative conversion without or withmercaptoethanol

The cloning of the gene from Pseudomonas and the expression in E. coilhas been described, for example, in EP-P-0504798 (U.S. Pat. No.5,830,743) and EP-A-0708180 (U.S. Pat. No. 5,766,881). The use ofmicroorganisms or enzymes of these for preparing 7-ACS is disclosed inEP-A-0275901 (U.S. Pat. No. 4,990,444) and EP 0525861 B1 (U.S. Pat. No.5,332,663). A purification process for the enzyme, using anoverproducing E. coli strain, is described in D. Bianchi et al., Enzymeand Microbiological Technology 20 (1997) 368-372.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects as illustrative onlyand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

TABLE 1 Amino acid analysis protocol of GAE Radical Number Mole percentA = Ala 85 11.806  B = Asx  0 0.000 C = Cys  0 0.000 D = Asp 49 6.806 E= Glu 32 4.444 F = Phe 31 4.306 G = Gly 55 7.639 H = His 13 1.806 I =Ile 23 3.194 K = Lys 11 1.528 L = Leu 57 7.917 M = Met 14 1.944 N = Asn30 4.167 P = Pro 55 7.639 Q = Gln 34 4.722 R = Arg 52 7.222 S = Ser 375.139 T = Thr 45 6.250 V = Val 49 6.806 W = Trp 16 2.222 Y = Tyr 324.444 Z = Glx  0 0.000 A + G 140  19.444  S + T 82 11.389  D + E 8111.250  D + E + N + Q 145  20.139  H + K + R 76 10.556  D + E + H + K +R 157  21.806  I + L + M + V 143  19.861  F + W + Y 79 10.972  Sum forthe entire sequence: Molecular weight = 79685.47 Number of radicals =720 Mean molecular weight per radical = 110.674 Charge = −18 Isoelectricpoint = 5.20 Extinction coefficient = 132000

What is claimed is:
 1. A process for delaying the deactivation ofglutaryl amidase during catalysis by said amidase in a substratesolution, comprising bringing the glutaryl amidase into contact with atleast one thiol selected from the group consisting of 2-mercaptoethanoland cysteine.
 2. The process of claim 1, wherein the glutaryl amidase iscoupled to a polymeric support.
 3. The process of claim 2, wherein thepolymeric support is an oxirane-activated polyacrylate.
 4. The processof claim 1, wherein said at least one thiol is 2-mercaptoethanol.
 5. Theprocess of claim 4, wherein the concentration of 2-mercaptoethanol is inthe range from 1 to 100 mM.
 6. The process of claim 1, wherein said atleast one thiol is cysteine.
 7. The process of claim 6, wherein theconcentration of cysteine is in the range from 1 to 100 mM.
 8. Theprocess of claim 1, wherein, during the amidase catalysis, said at leastone thiol is continuously present in the substrate solution.
 9. Theprocess of claim 8, wherein the glutaryl amidase is removed from thesubstrate solution and then brought into contact with said at least onethiol.
 10. The process of claim 9, wherein the substrate of the amidasecatalysis is glutaryl-7-aminocephalosporic acid.
 11. The process ofclaim 10, wherein glutaryl-7-aminocephalosporic acid is in theconcentration range from 5 to 500 mM.
 12. A process for preparing7-aminocephalosporic acid, comprising a) reactingglutaryl-7-aminocephalosporic acid with glutaryl amidase; and b)delaying the deactivation of said glutaryl amidase by bringing saidglutaryl amidase into contact with at least one thiol selected from thegroup consisting of 2-mercaptoethanol and cysteine.